Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902)

Overview

  • Product name
    Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit
    See all Pyruvate dehydrogenase kits
  • Detection method
    Colorimetric
  • Sample type
    Cell culture extracts, Tissue Lysate, Purified mitochondria
  • Assay type
    Enzyme activity
  • Assay duration
    Multiple steps standard assay
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human
  • Product overview

    Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902) can be used to determine the activity of PDH in a human, bovine, mouse, or rat sample. The PDH enzyme is immunocaptured within the wells of the microplate and activity is determined by following the reduction of NAD+ to NADH, coupled to the reduction of a reporter dye to yield a colored reaction product with an increase in absorbance at 450 nm at room temperature. Included for performance of the activity assay are buffer, detergent, reagent mix, and a 96-well microplate with monoclonal antibody pre-bound to the wells of the plate, allowing for a stream-lined assay.


    This assay is optimized for use with whole tissue extract when the amount of total material available for assay is 20-100 µg or more. If using cell extract of cultured cells 1 mg of material is required due to the very low levels of enzyme and reduced levels of mitochondria in the extract.


    The PDH complex is relatively fragile and sensitive to detergent. Please follow the sample preparation steps provided in the protocol booklet. Other preparation methods may decrease PDH activity. Our Scientific Support team is happy to answer any questions about sample prep.


    Review our Metabolism Assay Guide to learn about assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also about how to assay metabolic function in live cells using your plate reader.

  • Notes

    Store 20X Buffer, Detergent, and Microplate at 4°C.

    Store 5X Stabilizer at -20°C.

    Store 20X Reagent Mix, 100X Reagent Dye and 100X Coupler at -80°C.

  • Tested applications
    Suitable for: Functional Studiesmore details
  • Platform
    Microplate

Properties

Applications

Our Abpromise guarantee covers the use of ab109902 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Functional Studies Use at an assay dependent dilution.

Images

  • Mitochondria, tissue extracts and whole cultured cell extracts all show linear relationships between signal and sample load at limiting concentrations. The rates shown were determined as change in OD over time, and these are best represented as change in milliOD per minute.
  • Example of microplate reader recorded data from bovine heart mitochondria (100 µg/well) (top trace) and 2-fold dilutions (stepwise lower traces) using Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902).
  • Schematic diagram showing the reaction involved in Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902).
  • Principle of Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902)

Protocols

References

This product has been referenced in:
  • Nguyen QL  et al. Platelets from pulmonary hypertension patients show increased mitochondrial reserve capacity. JCI Insight 2:e91415 (2017). Read more (PubMed: 28289721) »
  • Zhang Y  et al. Induction of Posttranslational Modifications of Mitochondrial Proteins by ATP Contributes to Negative Regulation of Mitochondrial Function. PLoS One 11:e0150454 (2016). Functional Studies ; Mouse . Read more (PubMed: 26930489) »

See all 24 Publications for this product

Customer reviews and Q&As

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Pyruvate dehydrogenase activity in HCT116 cells

Average Average 3/5 (Ease of Use)
Abreviews
We wanted to analyse the enzymatic activity of pyruvate dehydrogenase (PDH) in human HCT116 cells dependent on the glucose level within the media. Therefore we cultivated these cells in DMEM with high (4.5 g/l) and low (1 g/l) glucose for 48 h. They were harvested and lysed in PBS. We used whole cell lysates to purify proteins. The PDH activity was spectroscopically measured by a change in absorbance at 450 nm. The absorbance was measured every minute for 30 min and kinetics were analysed with GraFit. The changed absorbance per minute was plotted against protein mass used to load the plate (fig. A and B). The blank value was calculated from four blanks in each test.
Unfortunately we only obtained significant activities at 500 µg protein or higher (fig. A). Thus we repeated the test with higher protein concentrations (fig. B). But in both test, no substantial difference between cells cultivated in high or low glucose was observed.
For these assays we had the problem that the four blanks in each test had a high inter-plate variation. This may be due to our plate reader or to the plate delivered with the assay kit. Therefore you should always use technical replicates. Additionally we think that problems can easily appear if the protein quantification during the test is incorrect and accordingly the loading of the plate.
In general the assay kit is not easy to use but gives a quick method to analyse the PDH activity.
Username

Mr. Christian Marx

Verified customer

Submitted Oct 04 2013

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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